This project will be carried out in Argentina as an expension of the parent grant DK56598 (4/15/99-3/31/03). Glycerol phosphate acyltransferase (GPAT) catalyzes the first and committed step in glycerolipid synthesis, the acylation of the sn-glycerol-3-phosphate to lysophosphatidic acid. We previously demonstrated that the mitochondrial isoform of GPAT (mitoGPAT) directs the cellular synthesis of triacylglycerol (TAG), but not phospholipids, in two different cell lines, suggesting that mitoGPAT may be a good candidate for the development of drugs to control TAG synthesis in the pathological conditions of obesity and diabetes. MitoGPAT is an outer mitochondrial membrane protein with two transmembrane domains. The protein contains an active site in the cytosolic N-terminal domain, a 84 amino acid loop that faces the mitochondrial intermembrane space, and a 238 amino acid C-terminal domain that has no known function. Our previous results showed that the enzyme is inactivated by inserting an epitope tag in the loop or by truncating at the end of the loop region. We propose to determine how mitoGPAT activity is regulated by the loop and C-terminal domains which do not form the catalytic site. The proposed experiments will analyze the activity of different truncated and mutated mitoGPATs expressed in CHO cells. We will determine which fragments of the mitoGPAT are essential for its proper function. We will also determine possible protein-protein or intraprotein interactions that regulate the enzyme activity by co-expressing in CHO cells mitoGPATs constructs tagged with different epitopes. The membrane fractions of the cells co-expressing mitoGPATs (typically the full-length active one and a modified-inactive one, or two full-length active proteins tagged with different epitopes) will be cross-linked and probed for the epitopes expressed with each protein. Thus, we will be able to elucidate whether the physical contact of mitoGPAT in the membrane, either with other specific proteins or with functional oligomers, is prevented in the modified proteins. These results will elucidate the regulation of the pathway of triacylglycerol synthesis, and may suggest novel strategies for enzyme inhibition, different from those targeted to mitoGPAT's active site.